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A Cascade of Arabinosyltransferases Controls Shoot Meristem Size in Tomato

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A Cascade of Arabinosyltransferases Controls Shoot Meristem Size in Tomato ARTICLES A cascade of arabinosyltransferases controls shoot meristem size in tomato Cao Xu1,9, Katie L Liberatore1,2,8,9, Cora A MacAlister1,8, Zejun Huang3,8, Yi-Hsuan Chu3, Ke Jiang1,8, Christopher Brooks1, Mari Ogawa-Ohnishi4, Guangyan Xiong5,8, Markus Pauly5,6, Joyce Van Eck7, Yoshikatsu Matsubayashi4, Esther van der Knaap3 & Zachary B Lippman1,2 Shoot meristems of plants are composed of stem cells that are continuously replenished through a classical feedback circuit involving the homeobox WUSCHEL (WUS) gene and the CLAVATA (CLV) gene signaling pathway. In CLV signaling, the CLV1 receptor complex is bound by CLV3, a secreted peptide modified with sugars. However, the pathway responsible for modifying CLV3 and its relevance for CLV signaling are unknown. Here we show that tomato inflorescence branching mutants with extra flower and fruit organs due to enlarged meristems are defective in arabinosyltransferase genes. The most extreme mutant is disrupted in a hydroxyproline O-arabinosyltransferase and can be rescued with arabinosylated CLV3. Weaker mutants are defective in arabinosyltransferases that extend arabinose chains, indicating that CLV3 must be fully arabinosylated to maintain meristem size. Finally, we show that a mutation in CLV3 increased fruit size during domestication. Our findings uncover a new layer of complexity in the control of plant stem cell proliferation. A wide range of inflorescence architectures exist among closely phenotypes in maize and rice have shown that the CLV pathway related plants1, and this variation can be explained in part by the is conserved in dicots and monocots12. However, little is known rate at which meristems terminate in flowers and initiate new branch about the genes and molecular mechanisms underlying stem cell meristems from which new flowers will form2,3. However, less appre- proliferation in other crops, leaving unanswered whether the CLV Nature America, Inc. All rights reserved. America, Inc. Nature 5 ciated is the role of meristem size in shoot architecture, as well as pathway or other pathways are important in controlling meristem in flower and fruit production. In maize, for example, variation in size and productivity. Here we investigate the control of meristem © 201 meristem size can account for differences in kernel row number size in tomato. between inbred lines4, and high row number is a major driver of yield in cultivated hybrids5. Meristem size is controlled by a main- RESULTS tenance mechanism that replenishes stem cells lost to lateral organ Tomato fab and fin mutants have enlarged meristems npg formation6. In Arabidopsis thaliana, meristem maintenance is based We explored the genetic control of inflorescence architecture in on a feedback system comprising the stem cell–promoting WUS tomato by screening a large ethyl methanesulfonate (EMS) and fast homeodomain transcription factor and CLV signaling proteins. CLV neutron (FN) mutagenesis population for mutants with increased signaling involves binding of the glycopeptide CLV3 to cell surface inflorescence branching13. In contrast to previously characterized leucine-rich repeat (LRR) receptor complexes that include the recep- mutants in which unbranched wild-type inflorescences are trans- tor kinase CLV1 and related LRR receptors7–10. These interactions formed into highly branched structures with normal flowers as a result initiate a signaling cascade that restricts WUS expression to prevent of delay in meristem maturation3,14, we identified six new mutants stem cell overproliferation, and, through negative feedback, WUS where branching was associated with fasciated flowers having more promotes CLV3 expression to limit its own activity11. Mutations floral organs than wild-type flowers (Fig. 1a–h). Complementation in CLV pathway genes cause meristems to enlarge, which can lead tests identified two loci that we designated fasciated and branched to increased shoot and inflorescence branching, more flowers, and (fab: one EMS allele) and fasciated inflorescence (fin: two EMS and extra organs in flowers and fruits. Studies on mutants with these three FN alleles). 1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA. 2Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA. 3Department of Horticulture and Crop Science, Ohio State University, Wooster, Ohio, USA. 4Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa, Nagoya, Japan. 5Energy Biosciences Institute, University of California, Berkeley, Berkeley, California, USA. 6Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California, USA. 7Boyce Thompson Institute for Plant Science, Ithaca, New York, USA. 8Present addresses: Cereal Disease Laboratory, US Department of Agriculture, Agriculture Research Service, St. Paul, Minnesota, USA (K.L.L.), Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA (C.A.M.), Chinese Academy of Agricultural Sciences, Institute of Vegetables and Flowers, Beijing, China (Z.H.), Dow AgroSciences, LLC, Indianapolis, Indiana, USA (K.J.) and Department of Anatomical Sciences and Neurobiology, University of Louisville, Louisville, Kentucky, USA (G.X.). 9These authors contributed equally to this work. Correspondence should be addressed to Z.B.L. ([email protected]). Received 22 February; accepted 27 April; published online 25 May 2015; doi:10.1038/ng.3309 784 VOLUME 47 | NUMBER 7 | JULY 2015 NATURE GENETICS ARTICLES Figure 1 The fab and fin mutants develop a b c d branched inflorescences with fasciated flowers WT fab fin fin as a consequence of enlarged meristems. (a) An unbranched inflorescence typical of a wild-type (WT) plant and a typical wild-type flower (inset). (b,c) Primary inflorescences from fab (b) and fin (c) mutants showing branching 1 1 1 8 2 11 and fasciated flowers (insets). (d) A branched 5 2 3 7 3 inflorescence from a fin side shoot. Red 4 9 5 4 3 6 5 7 Side-shoot inorescence arrowheads, branches; blue arrowheads, extra side shoots. (e–g) Wild-type fruits produce two e f g h locules (e), whereas the fruits of fab (f) and fin (g) WT fab fin WT fab fin mutants develop extra locules. Arrowheads, *** 16 *** locules. (h) Quantification and comparison of 14 *** *** 12 *** floral organ numbers in wild-type, fab and fin 10 *** *** flowers. (i–k) Stereoscope images of the primary 8 *** 6 SAM from wild-type (i), fab (j) and fin (k) plants 4 2 at the transition meristem (TM) stage, before Floral organ number 0 formation of the first flower. Dashed lines mark Sepal Petal Stamen Carpel the width and height used to measure meristem size. L8, leaf 8. (l) Quantification of TM size i j k l from wild-type, fab and fin plants. Data are WT fab fin 400 *** m) L8 µ means ± s.d.; n = 25 (h) and 9–13 (l). A two- 300 *** *** L8 L8 tailed, two-sample t test was performed, and 200 *** significant differences are represented by black asterisks: ***P < 0.0001. Scale bars: 1 cm (a–g) 100 and 100 µm (i–k). Meristem size ( 0 Width Height Inflorescence branching in fin mutants was more severe than in and Supplementary Table 1). Notably, there was significant overlap fab mutants, and fin plants also developed a highly fasciated primary (P < 0.001) among the differentially expressed genes (Fig. 2a), sug- shoot with more side shoots than wild-type plants (Fig. 1a–d). Both gesting that the FAB and FIN genes could act in the same pathway. mutants produced larger fruits as a consequence of additional carpels, Because fab and fin meristems were enlarged, we checked for expres- with fin mutants bearing the largest fruits (Fig. 1e–g). We designated a sion changes in the homolog of WUS (SlWUS, Solyc02g083950)16,17 strong allele of fin as a reference (fin-e4489, hereafter fin unless other- and a likely functional homolog of CLV3 (SlCLV3, Solyc11g071380) wise noted), and quantification of floral organs in the corresponding (Supplementary Fig. 4a–c)18. Despite a 25-fold increase in SlCLV3 Nature America, Inc. All rights reserved. America, Inc. Nature plants confirmed greater fasciation than in fab plants (Fig. 1h). expression, SlWUS expression doubled in fin meristems (Fig. 2b 5 Analyses of double mutants of fab and fin with the meristem matura- and Supplementary Table 1). CLV-WUS feedback was also mildly tion mutants compound inflorescence (s) and anantha (an), defective affected in fab mutants: SlCLV3 expression was upregulated by © 201 in the homolog of Arabidopsis WUSCHEL-RELATED HOMEOBOX 9 nearly fivefold, but SlWUS expression was maintained, consistent and the floral-identity gene UNUSUAL FLORAL ORGANS, respec- with a minor increase in vegetative meristem size (Supplementary tively, showed additive genetic relationships, indicating that the genes Fig. 2a–d and Supplementary Table 1). Notably, two additional npg underlying fin and fab act separately from the meristem maturation CLV3/EMBRYO-SURROUNDING REGION (CLE) genes (SlCLE3, pathway (Supplementary Fig. 1)14. Solyc02g067550; SlCLE9, Solyc06g074060) were also upregulated The phenotypes of fab and fin mutants were reminiscent of CLV in fin mutants (Fig. 2b and Supplementary Fig. 4d,e)18. We vali- gene mutants in Arabidopsis, in which inflorescence and flower fascia- dated the expression changes for SlWUS and SlCLV3 using in situ tion are caused by enlarged shoot apical meristems (SAMs)7,8,10,15. hybridization, which further showed substantial expansion of their The vegetative SAMs of the fab and fin mutants, unlike those of the
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